T cells play a pivotal role in anti-tumor responses, in the development of immune tolerance to self, and in autoimmunity. Cytotoxic T Lymphocyte-Associated antigen 4 (CTLA-4) is a surface receptor on T lymphocytes that down-regulates pathways of T-cell activation (Melero I, et al. Nat Rev Cancer. 7:95-106 (2007)), serving as an immune check point molecule. It is expressed intracellularly in resting T cells, transported to the T cell surface after activation of the T cell receptor (TCR). TCR engagement leads to tyrosine phosphorylation of CTLA-4 via the SRC kinase and releases it from AP50, resulting in its surface expression within 48 hours of T cell activation, leading to T cell tolerance and anergy. CTLA-4 expression is associated with decreased proliferation with cell cycle arrest at the G1-S interface and diminished cytokine secretion (Chambers C A, et al. Annu Rev Immunol. 19:565-94 (2001); Greenwald R J, et al. Immunity. 14:145-55 (2001)). It decreases cell proliferation through the inhibition of mitogen-activated (MAP) kinases but promotes T cell survival through the binding of phosphoinositol-3 kinase and activating protein kinase B (PKB/AKT) resulting in T cell anergy and tolerance without T cell death (Schneider H, et al. PLoS One. 3:e3842 (2008)). CTLA-4 signals suppress both CD4+ and CD8+ T cell responses via a tyrosine-based inhibitory motif (Egen J G, et al. Immunity. 16:23-35 (2002); Egen J G, et al. Nat Immunol. 3:611-8 (2002)).
CTLA-4 blockade has antitumor activity in mice, and important effects on breaking tolerance (Egen J G, et al. Nat Immunol. 3:611-8 (2002); Leach D R, et al. Science. 271:1734-6 (1996); Quezada S A, et al. J Clin Invest. 116:1935-45 (2006); Chen L, et al. Cell. 71:1093-102 (1992); van Elsas A, et al. J Exp Med. 190:355-66 (1999)). In experiments with B16 melanoma, a therapeutic effect induced by CTLA-4 blockade with a vaccine was associated with development of autoimmune vitiligo, suggesting that expansion of T cells recognizing melanocyte lineage antigens was associated with the therapeutic effect. Several autoimmune diseases were associated with single nucleotide polymorphisms in the CTLA-4 gene, including hypothyroidism and type 1 diabetes (Ueda H, et al. Nature. 423:506-11 (2003)). In CTLA-4−/− knock-out mice, expansion of lymphocytes with diffuse lymphadenopathy and lymphoid infiltration of different organs occurs, consistent with a generalized expansion of T cells (Waterhouse P, et al. Science. 270:985-8 (1995)). Similarly, both preclinical and clinical data indicate that CTLA-4 blockade results in activation and expansion of total CD4+ and CD8+ effector T cells (Wolchok J D, et al. Oncologist. 13 Suppl 4:2-9 (2008)), and breaking of self-tolerance has been shown in patients, as evidenced by the occurrence of immune-related adverse events (irAEs) observed with two different CTLA-4 antibodies, ipilimumab (Bristol Myers Squibb, Princeton, N.J., USA) (Maker A V, et al. J Immunol. 175:7746-54 (2005)) and tremelimumab (Pfizer, New York, N.Y., USA) (Ribas A, et al. Oncologist. 13 Suppl 4:10-5 (2008)).
Ipilimumab, a fully human, CTLA-4 blocking IgG1 monoclonal antibody induces long-lasting clinical responses in a minority of patients with metastatic melanoma (Wolchok J D, et al. Lancet Oncol. 11:155-64 (2008); Weber J, et al. Clin Cancer Res. 15:5591-8 (2009); Weber J. Cancer Immunol Immunother. 58:823-30 (2009); Fong L, et al. J Clin Oncol. 26:5275-83 (2008); Phan G Q, et al. Proc Natl Acad Sci USA. 100:8372-7 (2003); O'Day S J, et al. Ann Oncol. 21:1712-7 (2010)). Ipilimumab, with or without a gp100 peptide vaccine, compared with gp100 vaccine alone, improved overall survival (OS) in patients with previously treated metastatic melanoma (Hodi F S, et al. N Engl J Med. 363:711-23 (2010)). Ipilimumab combined with dacarbazine improved overall survival in previously untreated patients compared to dacarbazine alone (Robert C, et al. N Engl J Med. 364:2517-26 (2011)). These were the first randomized Phase III trials to demonstrate a significant survival impact for patients with metastatic melanoma, yet few studies have documented pharmacodynamic markers of the impact of ipilimumab. An increase in the absolute lymphocyte count (ALC) after 2 or 3 doses of the drug at weeks 4 and 7 has been documented (Yuan J, et al. Cancer Immun. 10:1 (2010)), and may correlate with an improved outcome; increased CD4+ HLA-DR+ T cells have been shown in several studies to occur after ipilimumab therapy (Sanderson K, et al. J Clin Oncol. 23:741-50 (2005); Sarnaik A A, et al. Clin Cancer Res. 17(4):896-906 (2010)); in several small cohort studies of brief duration, ipilimumab treatment increased the frequency of CD4+ICOShi T cells in tumors and in the circulation, and also induced antibody reactivity against the cancer-testis antigen NY-ESO-1 Carthon B C, et al. Clin Cancer Res. 16:2861-71 (2010); Chen H, et al. Proc Natl Acad Sci USA. 106:2729-34 (2009)). CTLA-4 abrogating antibodies do not alter vaccine specific immune responses (Attia P, et al. J Clin Oncol. 23:6043-53 (2005)) and even when administered with a peptide vaccine, tumor antigen specific responses were only modestly increased (Attia P, et al. J Clin Oncol. 23:6043-53 (2005); Ribas A, et al. Clin Cancer Res. 15:390-9 (2009)). Recall responses to viral and other antigens were not altered by ipilimumab. In patients receiving another CTLA-4 abrogating antibody, tremelimumab, the ratio of intratumoral T cells to FoxP3 positive T regulatory cells was increased after treatment only in regressing lesions (Ribas A, et al. Clin Cancer Res. 15:390-9 (2009)), suggesting a therapeutic impact of CTLA-4 abrogation on T cells infiltrating the tumor. The same investigators also demonstrated that peripheral blood Th17 cells were induced by tremelimumab (von Euw E, et al. J Transl Med. 7:35 (2009)), and that certain signaling pathways downstream of the TCR and cytokine receptor were also influenced by CTLA-4 blockade, such as increased pp38, pSTAT1 and pSTAT3, and decreased pLck, pERK1/2 and pSTAT5 levels (Comin-Anduix B, et al. PLoS One. 5:e12711 (2010)). CTLA-4 blockade also induced cell proliferation in the spleen, a secondary lymphoid organ, shown by molecular imaging with the PET probe 18F-FLT (Ribas A, et al. J Nucl Med. 51:340-6 (2010)). They also reported significantly increased intratumoral CD8+ T cell infiltration and CD4+ T cells infiltration, demonstrated the activation of lymphocytes within tumor sites, as increase of HLA-DR and CD45RO double positive cells in post tremelimumab biopsies (Huang R R, et al. Clin Cancer Res. 17:4101-9 (2011)) and increased expression of FoxP3.
To date, the precise molecular basis and mechanisms of action of ipilimumab have not been documented systematically in vivo. There is a critical need for biomarkers of the effects of ipilimumab as well as predictive biomarkers for clinical outcome and induction of irAE.
PD-1 blocking antibody (BMS-936558) therapy has shown antitumor activity and clinical benefit in melanoma patients. The precise molecular basis and mechanisms of PD-1 blockade in vivo have not been documented and there are few biomarker associated with clinical benefit. There is a critical need for biomarkers of the effects of PD-1 blockade treatment as well as predictive biomarkers for clinical outcome.